Establishment And Application Of Indirect Competitive ELISA Method For Chlordimeform Detection

Chlordimeform is one of the potent insecticides.It is extremely toxic and it can threaten personal health and life safety through the food chain.The metabolites in it are highly carcinogenic.This class of pesticides is listed as a banned drug by the international community.The purpose of this study was to establish an enzyme-linked immunosorbent assay for the detection of insecticidal mites.In the detection of insecticidal cockroaches,the Chlordimeform derivative was first synthesized by thionyl chloride method,the insecticidal cockroach artificial antigen was synthesized by succinic anhydride method,and the small molecule of insecticidal cockroach and carrier protein OVA and BSA were firstly treated by succinic anhydride method.Coupling,using Chlordimeform-BSA as immunogen,immunizing animals,preparing high-affinity,high-specificity antibodies,using ProteinA agarose gel affinity chromatography to purify antiserum,an indirect competitive ELISA method for the detection of insecticidal mites was established with optimization of coating time,blocking time optimization,blocking solution,optimization of optimal antigen and antibody working concentration,optimization of competitive reaction time,optimization of secondary antibody dilution,etc.The indirect competitive ELISA method established in this study has good performance of specificity and sensitivity.The IC50(Semi-inhibition rate)of the prepared insecticidal polyclonal antibody was3.176 ng/ml,IC90(the minimum detection limit)is 0.1148ng/ml,and the optimized condition was 4 ° C for 16 h,5% skim milk powder was blocked for 2 h,the optimal coating concentration of the antigen was 16 ug/ml,and the optimal working concentration of the primary antibody was 1 :25600,the competitive reaction time is2 h,and the secondary antibody dilution is 1:3000;this optimization is applied to the establishment of an indirect competitive ELISA method.The recovery rate of the method is between 83% and 117%,the sensitivity is 0.1ng/ml,and the coefficient of variation is CV < 20%.The method established in this study successfully determined the content of insecticidal mites in the water samples of the turbot artificial culture center in Xingcheng City,Liaoning Province.And established the curve under the water sample background.Compared with other detection methods,the method is simple,short-time,and high-specific.The method isapplied to detect the simulated water samples of the turbot culture center of Xingcheng City,Liaoning Province,which provides a certain theory and practice for the detection technology of insecticides basis.The research of this subject explored the rapid detection method of insecticidal cockroaches.By synthesizing special artificial antigens,specific polyclonal antibodies were prepared,and an indirect competitive ELISA method was established.In addition,this study used a water sample containing insecticidal mites as an analytical sample,and its high detection rate also indicated the necessity of studying the rapid detection method of chlorpyrifos.The established method can roughly reduce the workload to a certain extent without accuracy,and can detect water samples quickly,efficiently and at low cost,and further promote the rapid detection method of illegal drugs in aquatic products.To lay a certain foundation.